Ann. Re,·. NII/T. /984.4:339-76

NUTRITION ANDBIOCHEMISTRY OF TRANSAND POSITIONAL FATTYACID ISOMERS INHYDROGENATED OILS l

E. A. Emken

Northern Regional Research Center. Agricultural Research Service. U.S. Departmentof Agriculture. Peoria. Illinois 61604

CONTENTS

INTRODUCTION..................................................................................... 340

DIETARY SOURCES OF ISOMERIC FATTY ACIDS 340

DIETARY INTAKE OF HYDROGENATED FATS AND FATTY ACID ISOMERS... 341

PHYSIOLOGICAL AND METABOLIC STUDIES 342Absorption.......................................................................................... 342Growth. Organ Size,and Longevity......... 343Cell Function 344

EFFECT OF ISOMERIC FATS ON LIPID-METABOLIZING ENZYMES 345Desaturases and Elongases 345Lecithin:Cholesterol Acyltransferase 346Oxygenases......................................................................................... 346Prostaglandin Synthetase........................................................................ 347

EFFECT OF ISOMERIC FATS ON LIPOPROTEINS. TISSUE LIPIDS. AND FATTYACID COMPOSITION................................................................. 347

Serum Lipid and Lipoprotein Concentrations 347Tissue Lipid Class Concentrations............................................................. 348Farry Acid Composition 349

HEALTH-RELATED NUTRITION STUDIES 350Atherosclerosis.................................................................................... 350Cancer 351Prostaglandins and Related Studies........................................................... 352

lThe US Government has the right to retain a nonexclusive royalty-free license in and to anycopyright covering this paper.

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INCORPORATION. DISTRIBUTION. AND TURNOVER OF FATrY ACID ISOMERS 353MOlloelle Isomers 354Dielle Isomers 357

IN VITRO SPECIFICITIES OF ENZYME SYSTEMS FOR ISOMERIC FATS.......... 358Lipases 358ACYl CoA SmThelllse 358o.ridarioll 359ReTrocOIl\'ersioll . 361Phospholipid AC.I'lTrallsferase 361CholeSTerol EsTerase alld H.I'Clrolase........................................................... 363DesaTurases alld Elollf(ases 364

INTRODUCTION

Knowledge of the nutritional importance of dietary fats has greatly expandedsince the time when fats were considered only a source of calories. We nowknow that dietary fats supply the essential fatty acids (linoleic and linolenicacid) that are precursors for prostaglandins and that they are important compo­nents of membrane structures. Fats also influence cell function, serve ascarriers for fat-soluble vitamins, affect immunological function. and are associ­ated or involved with a number of diseases and disorders.

We also know that hydrogenated fats contain many fatty acid isomers notpresent in unhydrogenated oils and that in 1975. 5.6 billion Ib of hydrogenatedvegetable oil were produced in the United States, which is an average of 28Ib/year/person (113).

These facts explain why food technologists, nutritionists, biochemists. andthe medical community are interested in the nutritional value of hydrogenatedvegetable oils. The presence of isomers in hydrogenated fats has causedconsiderable controversy and has prompted a number of reviews. books.symposia, and special committee reports (10,23,38,53-55.57,70,90,93,107, II I, 116, 172,211. 213).

The purpose of this chapter is to summarize a variety of nutritional, biochem­ical, and related physiological data from animal and human studies that involveboth hydrogenated vegetable oil and specific fatty acid isomers.

DIETARY SOURCES OF ISOMERIC FATTY ACIDS

The process of catalytic hydrogenation of vegetable oils was discovered in1897 and is now extensively used to convert liquid oils to semisolid fats, whichfacilitates their formulation into margarines and shortenings. Partial hydrogen­ation also reduces the polyunsaturated fatty acid content of vegetable oils,which improves flavor stability and increases the length of time that cookingoils can be used in deep-fat-frying operations.

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An important side reaction of catalytic hydrogenation is isomerization,which forms more than 20 trans (t) and cis (c) positional fatty acid isomers (28,161, 183). The percentage of isomers in hydrogenated vegetable oil varieswidely with hydrogenation conditions (15, 27,28,79,93,148, 161,180,183,220), but typical hydrogenated soybean oil products contain about 12% t-18: 1,4% c-18:1, and 9% 18:2 and 18:3 positional and geometrical isomers. In theUnited States, hydrogenated soybean oil (HSBO) is the primary dietary sourceof fatty acid isomers, because about 90% of the hydrogenated vegetable oilproduced is HSBO (114). HSBO is used in a variety of commercial foodproducts other than margarines, shortenings, salad oil, and cooking oil. Theseinclude breads, cakes, pies, rolls, candies, cookies, crackers, mayonnaise,potato chips, roasted nuts, and many other snack and convenience foods. Infact, use of HSBO in processed foods is so widespread that it is nearlyimpossible to avoid consuming it.

In addition to hydrogenated vegetable oil, milk and ruminant fats alsocontribute to dietary isomeric fatty acids. Of the animal fats, only ruminant fatsnormally contain fatty acid isomers, which are formed primarily by biohy­drogenation of linoleic and linolenic acid in the ruminant forestomach. Over400 different fatty acid isomers have been identified in milk fat, but most arepresent only in trace amounts (160). Milk fat has from 2 to 5% total trans fattyacid isomers, depending on the ruminant diet and how long it remains in theforestomach. For example, summer butter has a higher percentage of transisomers than winter butter (162) because of the higher percentage of roughagein the summer diet.

Hydrogenated marine oils (HMO) are a third source of dietary isomeric fats,in particular of isomers of the fatty acids with chains 20-22 carbons long. Theuse of fish and marine oils as edible oils is prohibited in the United States,although it is important in other countries. Nutritional studies related to HMOare not reviewed in this chapter.

DIETARY INTAKE OF HYDROGENATED FATS ANDFATTY ACID ISOMERS

US Department of Agriculture (USDA) agricultural statistical data for 1980estimated total production of animal and vegetable fats and oils for edible use at12.7 billion lb and total edible fat disappearance at 169 g/day/person (1).Approximately 44% of this was partially hydrogenated vegetable oil. Thenationwide food consumption survey of 1977-1978 (210) and the ten-state andHANES surveys (185) have shown that eating patterns vary considerably withage, sex, income, and life style. Fats and oils supplied about 40% of averagetotal calories, and hydrogenated vegetable oil is estimated to contribute about

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13.8% of this. Thus, hydrogenated vegetable oil is one of the more importantsources of calories in the US diet. In comparison, protein supplies 16% ofcalories and all carbohydrates, 43%.

Based on analyses of numerous hydrogenated oil samples and total fatconsumption, a weighted average has been used to calculate a daily averageintake of individual cis and trans positional monounsaturated isomers, assummarized in Table I (52, 176). Other estimates of the total dietary trans fattyacid intake for the United States, Sweden, and other populations have beenbased on production/disappearance statistics, dietary recall data, and the dupli­cate portion technique. In general, intake of dietary trans fatty acid isomers isestimated at 5-9 g/day per person, or about 3-6% of dietary fat and 2-3% oftotal energy intake (2, 60, 61, 63, 65, 80, 115).

Analysis of human adipose tissue provides an alternative method of estimat­ing dietary fatty acid isomer intake, because these isomers are not synthesizedby humans. Adipose tissue fatty acids have a relatively long half-life of400-600 days (39, 87), so adipose tissue reflects the long-term average ofdietary isomeric fatty acid content (17). Studies of United Kingdom, German,and US populations show a range of 3.8-9.2% total trans content in adiposetissue (80, 153, 205a), which is consistent with many of the estimates based onconsumption data. The distribution pattern for positional fatty acid isomers inthe adipose tissue of several US subjects clearly resembles the pattern normallypresent in partially hydrogenated soybean oil (151, 154). A correlation coeffi­cient of 0.97 for the distribution pattern of trans positional monoene isomers inadipose tissue was calculated by assuming that 90-95% of the isomeric fats inthe adipose tissue are contributed by HSBO, and the rest by butter fat.

PHYSIOLOGICAL AND METABOLIC STUDIES

AbsorptionThe "digestibility" or absorption of hydrogenated fats was first investigated inthe late 1920s, and in the early 1930s was extended to elaidic acid or "isooleicacid" (13, 195). These early rat studies and later investigations (156) indicated

Table 1 Estimated daily consumption of specific positional octadecenoic acid isomers'

Positional octadecenoic acid isomer (g)

6 7 8 9 10 11 12 13 14 Total

c-18:1 0.01 0.14 0.24 6.41 0.37 0.54 0.68 0.14 0.03 8.6t-18: 1 0.01 0.27 0.65 1.56 1.53 1.26 0.82 0.48 0.29 6.8

Total isomer content minus 9c-18:1 = 9.0 g

'Based on an estimated consumption of 34 g hydrogenated soybean oil containing 25% c·18:! and 20%t·18:\.

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that absorption of hydrogenated fat is equivalent to absorption of nonhydrogen­ated fats and oils. Chemically synthesized individual fatty acid isomers andtheir triglycerides have melting points that vary from - I3-54°C (14,34,66,72,73, 168, 169); recent animal studies with hydrogenated fats suggest that allisomers, including those with melting points higher than body temperature, areabsorbed as well as oleic acid (57, 120, 121, 225). These animal studies havebeen confirmed by human studies with pure triglycerides containing deuteratedcis and trans positional octadecenoic acid isomers (56, 58, 59); melting point,double bond position, and configuration have no measurable effect on theabsorption of any isomer.

These in vivo results agree with the data for in vitro pancreatic lipasehydrolysis of a series of triglycerides containing a single cis positionaloctadecenoic acid isomer, a saturated fatty acid, and linoleic acid (9c, 12c-18:2)(10 I). Triglycerides containing 18: I isomers with the double bonds in the 2 to 7positions were found to inhibit lipase hydrolysis of triglycerides, which isnecessary for fat absorption. The il8 to ill6 cis positional 18: I isomers, whichare the ones present in hydrogenated fats, did not inhibit pancreatic lipasehydrolysis; this data suggests that hydrogenated fats should be well absorbed.

Growth, Organ Size, and Longevity

Long term and multiple-generation studies with rats (4,5) and mice (214) fedpartially hydrogenated vegetable fats have been reported. Pathological ex­amination of organs showed no abnormalities. Growth, longevity, reproduc­tion, and lactation also were unaffected by diets containing 9.5% hydrogenatedoils. The trans content of the dietary fats fed was 35%, which was estimated tobe ten times the level in human diets. Growth curves of controls and of rats fed20% (by weight) HSBO containing 48% trans also showed no difference. Nochange in weight gain was reported when rats were fed 50 and 100 mg/day of a1:1 mix of9t-I8:1; c,t-18:2; or t,t-18:2 plus c,c-18:2 for 4 weeks (138). Theprotein efficiency ratio for casein was not influenced in rats fed diets containing7.8 or 20.1 % (by weight) HSBO, and even diets containing 7.8% trielaidin hadno nutritionally significant effect on protein utilization (98). In comparison to9c-18: I, which had no effect (200, 221), addition of 6c-18: I to rat novikoffhepatoma cell cultures reduced cell growth.

The only studies with hydrogenated fats that report lower growth rates andpathological abnormalities are those in which hydrogenated fats were fed toessential fatty acid-deficient (EFAD) rats (3, 89). In general, all fats except9c-18:1 and c,c-18:2 appear to accentuate EFAD, and 18:0; 9t-18:I;t,t-18:2;and c,t-18:2 produced the most significant results. The structures of t,t- andc,t-18:2 apparently are similar enough to that of c,c-18:2 that they compete inreacylation of membrane phospholipids. This competition would enhance theremoval or loss of the last remnants of c,c-18:2 and 20:4 from tissue. Other

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nonessential fats probably also promote this effect by a mass action or dilutionmechanism.

In contrast to animal studies, growth of bacteria (Escherichia coli) and yeast(Saccharomyces cerevisiae) cells was influenced by various positional 18:1isomers. The cell lines used were mutants not capable of fatty acid synthesis,and they were forced to utilize those fats added to the medium. Growth of E.coli and S. cerevisiae was inhibited by addition of ~8 to ~ 13 t-18: I positionalisomers even in the presence of added 9c-18: I (212). Growth inhibition was thegreatest for the 4t-, 6t-, 7t-, Ilt-, and 12t-18:1 isomers, which also promotedtriglyceride accumulation in the cells. Addition of 9t-18: I was reported todepress phospholipid synthesis and was considered to be the reason cells failedto divide (71). A more recent study reported normal cell growth with 9t- andIIt-I8: 1 when higher levels of adenosine monophosphate (AMP) were added.This suggests that trans acids provide sufficient membrane fluidity for cellgrowth and that they are involved in regulation of cell function (207).

In a related study with S. cerevisiae, the effects of a variety of otherunsaturated 20- and 22-carbon fatty acids on growth were compared to those ofthe~2to~15t-18:1series. The2t-t04t-and 13t-to 15t-18:1 isomers did notsupport growth, but 20:4 and 20:5 were twice as effective as 9c-18: I; 9c, 12c­18:2; or 9c,12c,15c-18:3 (216). In contrast to the t-18:1 isomers, the 7c- tolOc-18: 1 positional isomers were all about equally effective in supportinggrowth of E. coli and S. cerevisiae cells (152). The 14c- to 17c-18: I acids didnot support E. coli growth, and the 12c- to 17c-18: I acids did not support yeastcell growth. The 5c-18: 1 acid was effective for bacterial cell growth but not foryeast, and the 6c-18: 1 was effective for yeast cell growth but not for bacterialgrowth. No obvious relationships between physical properties of the fatty acidsand cell growth were noted.

Cell FunctionThe effects of various isomers on AMP requirements and growth of single cellorganisms described above support the general concept that membrane lipidsaffect the microenvironment of the cell and influence cell functions. Red bloodcell fragility data (41), swelling of cells in tissue culture (/32), and mitochon­dria permeability studies (35) indicate that membrane permeability and fluidityare altered by fatty acid isomers.

Other evidence that isomeric fats influence cell function involve the inhibi­tion of immunoglobulin interaction with lymphocytes by 9c-18: I; c,c-18:2; and20:4, but not by saturated fatty acids and 9t-18: I (78, 110). The difference ininhibition was associated with a change in membrane-bound calcium anddepletion of adenosine triphosphate (ATP) levels in lymphocytes, which wereattributed to uncoupled oxidative phosphorylation.

Rats fed hydrogenated fish oil for 15 weeks developed severe testicular

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degeneration (100), and a diet containing a 50:50 mixture t,t- and c,c-18:2reduced rat lung weights by 20% (231). Rats fed t,t-18:2 also had lowerfibrinogen concentrations, longer bleeding, and shorter prothrombin times thanrats fed com oil (167). Addition of 9t- and t,t-18:2 to rat heart cell cultureimmediately arrested pulsation or beating (arrhythmia) of the cells, whereas 9c­and c,c-18:2 had only mild effects (222).

These effects should not be viewed with particular alarm in terms of effectson humans, because the experimental designs employed produce environmentsmuch different than those encountered by cells in normal in vivo conditions. Nostudy of higher organisms using dietary fats similar to edible hydrogenatedvegetable oil has shown any discernible effect on cell or organ function.

EFFECT OF ISOMERIC FATS ON LIPID­METABOLIZING ENZYMES

A number of in vivo investigations have provided evidence that hydrogenatedfats and specific fatty acid isomers can influence the activity of the desaturases,elongases, acyltransferases, oxygenases, and prostaglandin synthetases.

Desaturases and ElongasesThis section summarizes a number of in vivo studies that provide evidence thathydrogenated fats, specific isomers, and nonisomeric fatty acid (18:3w3,20:3w6, and 20:4(6) inhibit desaturation and elongation of nonisomericpolyunsaturated fatty acids (209).

Rats fed 10 and 20% HSBO diets containing 49.5% t- and 1% c,c-18:2displayed an increased amount of 20:2w9 in liver phospholipids, which wasproduced by the low 18:2 levels in the diet rather than by trans fatty isomers.The level of 20:4 was also lowered, which implies reduction of desaturase­elongase activity because of isomeric fatty acids (46, 47) or because of the low18:2 levels in the diet. Others have reported that conversion of 9c-18: I and9c-16:1 to 20:3w9 and 20:4w7 was inhibited by addition of 9t- and t,t-18:2 tofat-free diets (166, 204), which also suggests reduction of desaturase-elongaseactivity. Rats fed an enriched cis and trans 18: I fraction from HSBO exhibitedreduced synthesis of 20:4 and accelerated synthesis of 20:5w3 and 20:3w9(126, 165). This effect was correlated with the 12c-18:1 isomer. A similarreduction in 20:4 levels has also been observed for plasma lipids from humansubjects fed mixtures of 9c-, 12t-, and 12c-18:1 (58).

Diets containing t,t-18:2 decreased the levels of 20:4 in rat liver and kidneylipids, which suggests that desaturation-elongation of 18:2 was inhibited (9,109). In similar studies, synthesis of 20:3 in rat lung was also inhibited (231).

Data from suckling rat brain injected with 14C-Iabeled 18:0, 9c-18:1, 9t­18:1, and t,t-18:2 indicated that t,t-18:2 and 9c-18:1 inhibited desaturation-

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elongation of c,c-18:2, while 9t-18: 1 and 18:0 stimulated desaturase-elongaseactivity (37). Studies of rats fed EFAD diets showed elevated ~6 desaturase­elongase activity, but results from rats fed EFAD diets containing 20% (byweight) of 9t- and t,t-18:2 indicated that t,t-18:2 suppressed the ~6 desatura­tion-elongation sequence (118). The effect of9t-18: 1 was considered minor. Invivo rat data showed that 6c-18: 1 inhibited conversion of9c-18: 1to 20:3w9 andconversion of l8:2w6 to 20:4w6, but diets containing 9c-18:1 had little effect(143). Feeding rats a mixture of 9t-18:1 and t,t-; t,c-; and c,t-18:2 isomersinhibited ~6 desaturase but not ~9 desaturase (42). Studies in which fat-freediets containing added c,c- and t,t-18:2 or c,c,c-18:3 and t,t-18:2 were fed torats demonstrated suppressed conversion of c,c-18:2 and c,c ,c-18:3 to longer­chain-length polyunsaturated fatty acids (188).

Rats fed a mixture of t, t- and c,c-18:2 at 5 and II % of calories had decreasedlevels of 20:3 and 20:4w6 fatty acids in heart, kidney, lung, platelets, andadipose tissue, which also suggested inhibition of the desaturase-elongasesystem (108). In contrast, conversion of 20:3w6 to 20:4w6 increased when ratswere fed HSBO (43% t-18:l, 2.4% t,c-18:2, and 16% c,c-18:2) for 28 and 43weeks, which suggests an increase in ~5 desaturase activity (84). Essentialfatty acid-deficient rats fed ethyl elaidate for 12 weeks exhibited an increasedrate for desaturation of 16:0 to 16:1 (74). Monoene isomers in hydrogenatedmarine oil produced lower 20:4 levels in rats due to competitive inhibition of~6 desaturase activity (91).

Lecithin:Cholesterol Acyl Transferase

Rats fed isomerized safflower oil containing 20.8% t-18:l and 47.5% t,t-18:2for 43 weeks showed an initial increase in lecithin:cholesterol acyl transferase(LCAT) activity, which decreased after 40 weeks (204). The depression ofLCAT activity was probably due to 9t,12t-18:l rather than 9t-18:1, based onother studies that indicated that LCAT and lipoprotein lipase activity did notchange when EFAD rats were fed 5% 9t-18:1; however, when 5% 9t,12t-18:2was added to the diet, LCAT and lipoprotein lipase activity decreased (165).Similarly, LCAT activity in liver from rats fed HSBO diets containing 12-50%total trans decreased as the percentage of trans increased (144).

Oxygenases

Respiratory activity of rat heart mitochondria was reduced when HSBO con­taining 48% t-18:1 and 3% 18:2 was fed for 6 weeks. These results suggestedthat t-18: 1 isomers decreased the activity of the (3-oxidation enzyme system(95). In a study related to oxidation of drugs, the activity of nicotinamide­adenine dinucleotide phosphate (NADP) cytochrome c reductase andcytochrome p-450 decreased in rats injected intraperitoneally with 9c, 12c­18:2; however, no effect was noted with 9t-18:1. Both fatty acids decreasedaryl hydrocarbon hydrolase and nitroanisole demethylase activity (83).

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Prostaglandin Synthetase

In vitro experiments with a limited number of polyunsaturated isomeric fattyacid structures suggest that conversion of 20:4 to prostaglandin (PG) is inhib­ited as a result of reduced cyclooxygenase activity. These and other studiesinvolving trans fatty acids and PG are summarized in a number of reviews (54,107, 137, 159).

When rats were fed a 1: 1 mixture of9t, 12t- and 9c, 12c-18:2, PGF2a levels inserum were 50% lower than in controls, but 20:4 levels were reduced by only12%. This result and an observed increase in susceptibility of platelet aggrega­tion to indomethcin for the 9t,12t-18:2 diet compared to a 9c,12c-18:2 dietimply that PG synthetase is inhibited by the t,t-18:2 isomer (96).

In vitTo data with bovine seminal vesicals was used to demonstrate inhibitionofPG synthetase by a number of 18- to 20-carbon-chain-Iength polyunsaturatedisomers (150). For the isomers studied, levels of inhibition were generally low,except when the isomer contained a .:l12t double bond conjugated with a .:l14cdouble bond. Inhibition was 74% for 8c,12t,14c-20:3 and 54% for5c,8c, 12t, 14c-20:4 (62). It is surprising that inhibition was not greater for theother isomers investigated, because 18:2 and 18:3 also inhibit PG synthetase(158).

In addition to the PG synthetase inhibition studies, a number of odd-chain­length cis polyunsaturated fatty acid (PDFA) isomers have been shown to beconverted in vitro to biologically active PG isomers (16); however, these arenot likely to be of nutritional importance.

EFFECT OF ISOMERIC FATS ON LIPOPROTEINS,TISSUE LIPIDS, AND FATTY ACID COMPOSITION

The influence of HSBO and specific fatty acid isomers on tissue, serum, andlipoprotein triglyceride (TG), phospholipids (PL), free fatty acids (FFA) ,cholesterol ester (CE), and cholesterol levels are related to cell function andenzyme activity. They are also related to the individual fatty acid compositionof the various lipid classes.

Serum Lipid and Lipoprotein ConcentrationsSeveral studies have investigated the effects of diets containing isomeric fats onserum lipid classes and lipoprotein levels in swine, rats, and rabbits. Resultshave been inconsistent, which may reflect variation in experimental design. Forexample, plasma CE, TG, PL, and cholesterol levels in swine fed HSBO (50%trans) were increased compared to levels in those fed corn oil diets, but theincrease in lipid classes was the result of low levels of 18:2 in the HSBO dietrather than of its isomer content (117, 230). Subsequent studies with swinereported that HSBO diets containing additional 18:2 did not significantly

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increase serum cholesterol, TG, and PL levels (49, 131). Lowering of high­density lipoprotein levels did occur when swine received diets containing transfatty acid isomers, but no changes in the physical properties of lipoproteinfractions were observed (99).

Studies of rats fed margarine containing hydrogenated rapeseed oil showedno effect on serum cholesterol and PL levels (232). In fact, rats fed variousHSBO diets containing 12-50% trans isomers were found to have lower serumcholesterol levels than rats fed com oil (144). In contrast, rats fed an isomerizedsafflower oil fraction containing 20.8% t-18: 1 and 47.5% t,t-18:2 for 43 weeksshowed increases in serum cholesterol ester and PL levels but lower TG levels.Little change in liver, kidney, and heart lipid class composition occurred (204).

Rats fed safflower oil and then switched to hydrogenated coconut oil or ahigh-trans diet containing 50% t,t-18:2 and 24% t-18:1 showed an immediatedecrease in high-density lipoprotein levels (181). An increase in low-densitylipoprotein levels occurred with threonine-imbalanced rats fed elaidinized oliveoil (187).

The most detailed study on the effect of hydrogenated vegetable fat on ratplasma lipid classes is that by Wood (225). Plasma TG, phosphatidylcholine(PC), FFA, and cholesterol concentrations increase after one week on thepartially hydrogenated safflower oil (PHSFO) diet and then gradually decreaseexcept for FFA. Other serum lipid levels (lysophosphatidylcholine, sphing­omyelin, and CE) did not change.

Rabbits fed diets containing hydrogenated olive oil and t,t- and c,t-18:2isomers from elaidinized safflower oil showed no significant changes in serumcholesterol and TG levels (127,189). In rabbits fed 9t-18:1, however, serumcholesterol levels doubled (139, 140,219).

The general impression is that isomeric fatty acids or hydrogenated vegetableoils have little effect on serum lipid class concentrations.

Tissue Lipid Class ConcentrationsThe effects offour different diets (HSBO, tallow, com oil, and fat-free) on CE,PL, and TG levels were reported for rat adrenal, liver, heart, and lipoproteinclasses (46). Comparison of a 20% (by weight) HSBO diet containing 48%trans and 1% 18:2 to a com oil diet fed for 15 and 20 weeks indicated thatHSBO significantly increased heart and very-low-density lipoprotein (VLDL)TG levels, reduced heart and high-density lipoprotein (HDL) PL levels, andincreased adrenal CE level at IS weeks. Effects of the diets on other tissue lipidlevels were not significant. Considerable variation in 15- and 20-week datasuggests that the low 18:2 level in the HSBO diet was probably a moreimportant factor at 20 weeks than were tissue types or trans isomer levels.

Livers from threonine-deficient rats fed olive and com oils contained 30%more lipid and lower low-density lipoprotein (LDL) levels than those rats fed

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elaidinized olive oil and corn oil (187). In threonine-imbalanced rats, feeding9t-18: I compared to 9c-18: I apparently prevented accumulation of liver lipidsby increasing the transport or mobilization of lipids from the liver to other sites(186). A hydrogenated fish oil diet that contained long-chain isomers wasreported to reduce TO levels by 50% in rat testicular lipids (100). Platelets fromrats fed t,t-18:2 contained more CE and TO and less PL than rats fed corn oil(167).

Detailed data are available on the concentration oflipid classes in livers fromrats fed partially hydrogenated safflower oil (225). The concentration of themajor PL classes was not influenced, but there were moderate increases in liverTG concentrations compared to those for chow-fed rats. Thus, existing dataindicate that except under extreme experimental conditions, hydrogenated fatshave little influence on tissue lipid class composition.

Fatty Acid CompositionMuch of the in vivo evidence that suggests that isomeric fatty acid altersdesaturase-elongase activity is based on changes that occur in fatty acidcomposition. Changes in tissue fatty acid composition can also result fromsimple mass or dilution effects and from biochemical control of the fatty acidcomposition of lipid classes. For example, in phosphatidylcholine there is thewell-known tendency for 16:0 to be paired with 20:4 in the same molecule.Triglycerides are also known to be esterified preferentially with unsaturatedfats at the 2-acyl position. Selective placement of fatty acids in these lipids isapparently due to the effect of lipid structure on membrane permeability andcell function.

The modification of fatty acid composition by dietary cis and trans positionalisomers in hydrogenated fats is complex. Modification of fatty acid composi­tion probably is the result of a combination of several factors, including enzymeactivity, enzyme selectivity, and dietary fat compositions. The effects of thesefactors vary with the tissue, specific lipid classes, and experimental conditions.The only generalization that can be made is that partially hydrogenated vege­table oil normally reduces 20:4w6 and increases 18: I levels.

Short-term human studies indicate that the positional octadecenoic acidisomers (9t-, 12t-, 12c-, 13t-, and 13c-) have different effects on plasma fattyacid composition (55, 56, 58, 59). In these studies, a single meal containingtriglycerides of one or two deuterium-labeled fatty acid isomers plus deuteratedoleic acid was fed, and fatty acid composition of plasma TO, CE, PC, andphosphatidylethanolamine (PE) were determined at maximum incorporation ofthe deuterated fats and compared to zero-hour data. Low levels (0.4---0.8%) ofthe fatty acid isomers incorporated into CE fractions had no effect on CE fattyacid composition. The relative percentage of the nonmonoenoic fatty acids inthe TO fraction was reduced because of the increase in 18: I from the meal fed.

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For plasma PC and PE, a decrease from 0.7 to 0.4 in the polyunsaturated!saturated (PIS) fatty acids ratio occurred when the 12c- and 12t-18:1 isomerswere fed. The change in the PIS ratio was the result of a large decrease in the20:4 percentage and a moderate increase in the 16:0 percentage. Analysis of the1- and 2-acyl PC fatty acids indicates that I2c-18: 1 is incorporated selectivelyinto the 2-acyl position, which is the reason for a decrease in polyunsaturatedfatty acids. In contrast, the 9t-18: 1 isomer did not decrease the PIS ratio of anylipid classes; however, the 13t- and 13c- I8: 1 positional isomers increased thePIS ratio from 0.6 to 0.9 in the PE fraction. These data suggest that each isomerhas a different effect on human serum lipid fatty acid composition, and thisconclusion is supported by rat data (20, 40,95, 108, 109). A comprehensivecompilation of fatty acid composition data is available for TO, PC, PE, SM,and phosphatidylserine (PS) from heart, kidney, liver, hepatoma, lung, mus­cle, spleen, and adipose tissue of PHSFO- and chow-fed rats (225).

One point that seems clear is that adipose tissue fatty acid compositionreflects the composition of dietary fat reasonably well and does not selectivelyincorporate or exclude any specific isomeric fatty acid.

HEALTH-RELATED NUTRITION STUDIES

A variety of diseases.and metabolic disorders involve fat metabolism (22, 50,51, 64). The more serious and common are coronary heart disease, cancer,multiple sclerosis, cystic fibrosis, and the variety of problems associated withor aggravated by obesity. Involvement of unhydrogenated dietary fats in theeitology of diseases is not well understood, and little is known about the effectsof hydrogenated fats and oils.

AtherosclerosisDietary fat is implicated as a factor in the etiology of atherosclerosis, butalthough large population studies have generated a wealth of information, thesestudies were not designed to differentiate between effects of hydrogenated andnonhydrogenated fats. For example, the National Diet-Heart Study completedin 1968 (6) used hydrogenated and unhydrogenated fats to vary saturate:mono­unsaturate:polyunsaturate ratios in the various diets. However, data was notobtained on the trans fatty acid content of the dietary fats or on blood lipidsamples.

At least 14 studies with smaller numbers of subjects have reported data thatcan be used to evaluate the effect of hydrogenated fats on blood lipids. Theresults of all but one of these studies (119) were reviewed in detail by Emken(57) and Applewhite (10). The subjects were students, prisoners, mentalpatients, monks, and nuns. Several different hydrogenated vegetable oils withiodine values of 55-109 and trans values of 10-70% were fed as dietary fats.

ISOMERIC FATTY ACIDS 351

Serum cholesterol levels were monitored, and in some experiments TG and PLlevels were also determined. Small increases (20-30 mg %) in serum cholester­01 levels as a result of isomeric fatty acids in hydrogenated fats were observed inabout half of the studies, and the others reported slight decreases. For thosestudies reporting serum TG and PL levels, the data were similar to thecholesterol data. The combined results indicate that replacement of hydrogen­ated fat with unhydrogenated fat in the diet is of little value in reducing serumlipid levels. Other factors such as total cholesterol intake, PIS ratio, general lifestyle, and genetic variability appear to have a greater influence on serum lipidsthan do dietary isomeric fatty acids.

Correlation of the trans fatty acid content of adipose tissue from UnitedKingdom subjects who died of heart disease and unrelated causes has beeninvestigated. Initially, no significant correlation of heart disease and trans fattyacid was reported (205a), but recently adipose tissue from heart disease victimswas found to contain higher levels of t-18:1 and t-16:l (205b).

Animal studies can use invasive techniques and dietary extremes not permit­ted in human studies. Animal studies that have investigated the effect of dietscontaining hydrogenated fat on atherosclerosis have been reviewed previously(10, 111, 213) and are briefly summarized here. The development of arteriallesions in swine fed HSBO (50% trans) containing little 18:2 correlated withpercent trans fatty acids (117), and as Applewhite pointed out (10), with low18:2 levels. Other studies with swine fed HSBO containing higher levels of18:2 vs unhydrogenated fats did not produce a higher incidence of fatty streaks,fibrous plaques, or lesions in the aorta and coronary artery than in swine fedunhydrogenated oil (49, 99, 131, 178).

Rabbit studies indicated trans fatty acids are hypercholesterolemic (139,140, 179, 219), but no effect on aortic atherosclerosis and no differences inliver enzyme activities were found. A recent study with Vervet monkeysdemonstrated no differences in serum or liver lipids or in the severity ofatherosclerosis (111). Rats fed a 10% hydrogenated corn oil (42% trans) dietshowed lower serum cholesterol levels, increased excretion of neutral andacidic steroids, and changes in steroid composition (201). Analysis of isolatedcardiac sarcolemma tissue from rats fed 5% 9t-18: 1 by weight showed mem­brane cholesterol content about 25% higher than that from rats fed 9c-18:1.Phospholipid levels were unaffected, with the 9t-18: 1 mostly incorporated intoPE and PC (12).

CancerThe scientific literature is replete with studies involving the effects of dietaryfat on tumor growth and development, but relatively few reports have specifi­cally focused on the effects of hydrogenated vegetable oils and specificisomeric fats. Reviews of numerous epidemiological studies report a positive

352 EMKEN

correlation between increased dietary fat intake and breast cancer (77 , 86, 102,142, 224, 229). Epidemiological data also suggest that PUFA are associatedwith a higher incidence of tumors than are saturated fats (92), and a positivecorrelation between hydrogenated vegetable oil and cancer has been reported(60, 61). Several studies have reported that high dietary fat levels enhancedevelopment of tumors induced by various carcinogens (177, 217), and PUFAwere found to promote tumors more effectively than saturated fats (29-31).

Others have critically reviewed the epidemiological data and the interpreta­tion of the results from animals fed high-fat diets and have concluded that thereis little evidence of a positive relationship between dietary fat and incidence ofcancer in humans (82). A dietary intervention study with Finnish men replacedsaturated fats in the diet with vegetable oil; they reported reduced mortalityfrom coronary heart disease but no increase in mortality due to malignancy(208).

A few experiments suggest that fatty acids have weak carcinogenic prop­erties. Data on the subcutaneous injection of mice with stearic, lauric, and oleicacids indicate that these fatty acids may be weak carcinogens (203). Mice fed20% (by weight) olive oil diets containing 50% trielaidin or trilinoelaidin werereported to have a higher incidence of liver tumors than did controls (106).These findings are questioned because a number of similar studies with miceand rats have not reported an increased incidence of tumors. Limited biochem­ical data is available on tumor cell metabolism offatty isomers. Ehrlich ascitestumor cells from mice fed elaidic acid incorporate about one half the percentageof 9t-18: 1 as normal liver cells and show reduced total lipid content (11). Alarge increase in the percentage of 18:0 also occurred in the tumor cells of micefed either 9c- or 9t-18: 1. These data suggest that the tumor cells had lost muchof their ability to regulate fatty acid composition of the cell lipids selectively,and the data are consistent with results published previously (227, 228). Incontrast, leukemia cells from mice do not lose their mechanisms for selectiveacylation of the 1- and 2-acyl positions of phosphatidylcholine and ethanola­mine with saturated and polyunsaturated fatty acids (26).

Prostaglandins and Related Studies

The potent biological activity of prostaglandin (PG) and leukotrienes on avariety of cell and tissue functions has stimulated a tremendous amount ofresearch. One reason is the interrelationship of PG and disorders such asatherosclerosis, hypertension, stroke; and platelet functions. For many years,researchers in prostaglandin biosynthesis and regulation have explored theimportance of 20:4w6 and 20:3w6 fatty acids, the precursors for a large numberof leukotriene and PG structures, and of linoleic acid, the precursor to 20:4 and20:3 fatty acids. More recently, the w3 fatty acids have been identified ashaving an important role in the regulation ofPG biosynthesis and physiologicaleffects of PG and PG-related structures (44). Dietary supplementation with w3

ISOMERIC FATTY ACIDS 353

fats has been reported to have significant physiological effects, especially onblood clotting, in spite of the relatively low levels of these fats in human tissue;there is little question that the amount of specific fatty acids in the diet caninfluence PG biosynthesis. As examples, the absolute amount of 18:2, ratherthan the percentage of 18:2 in the diet, appears to be responsible for increasedPG levels. Addition of20:4 to human diets increased the metabolite ofPGEz inurine by 47% and increased the reactivity of platelets (190). Consumption byhumans of 1-2 g of 20:3w6 increased 20:3w6 and PGE levels in plasma. Inrabbits, PGE I levels increased 20- to 30-fold and PGEz levels decreased by25-50% when 20:3w6 was added to the diet (44).

What effect do specific isomeric fats present in hydrogenated oils have on theratio and amounts ofPG in tissue? Relatively few specific isomers and fats havebeen studied in experiments designed to provide answers to this question.Much of the present knowledge on this subject has been covered in severalreviews (54, 88, 107, 137).

The studies from Kinsella's laboratory have centered around the relation ofthe t,t-18:2 isomer to PG functions and synthesis (24,96). In these studies, ratsfed diets containing equal amounts of t,t- and c,c-18:2 developed reducedresponse to platelet aggregating agents, which implied inhibition of PG synthe­sis by t,t-18:2. The t,t-18:2 diets also decreased levels of the w6 fatty acids(18:2,20:3, and 20:4) in heart, kidney, lung, platelet, and adipose tissues andreduced the levels ofPGE 1, PGEz, and PGFz in serum. In rats fed various levelsof t,t-18:2, hematological properties of red blood cells were affected at highlevels of t,t-18:2, thromboxane Bz levels were decreased, and 6­ketoprostaglandin Flex levels were unchanged. Little or no effect was found att,t-18:2 levels that correspond to those estimated present in human diets (24).The decrease in PG production by platelets was greater than the decrease in thefatty acid precursors, which suggests inhibition ofPG synthetase by the t,t-18:2isomer. Increased c,c-18:2 levels in serum PL suggested that conversion ofc,c-18:2 to 20:4 was inhibited by t,t-18:2, which confirmed earlier results byPrivett. However, Privett also reported that addition of c,c-18:2 to the dietnullified the effects of t,t-18:2 (165).

Other studies of rats fed hydrogenated fat reported higher PG levels than forfasted rats, which suggests that the additional dietary c,c-18:2 from hydrogen­ated fats more than compensates for PG synthetase inhibition by isomeric fats(159, 165).

INCORPORATION, DISTRIBUTION, AND TURNOVEROF FATTY ACID ISOMERS

Numerous studies have mapped the incorporation and distribution of isomericfats into tissue and lipid classes. The results of many of these have already beendiscussed in terms of the implied effect of isomeric fats on enzyme activities

354 EMKEN

and fatty acid composition, and they will be discussed later in tenus of enzymespecificity. The purpose of this section is to discuss the levels of isomeric fats inthe diet that are necessary to influence the isomeric fatty acid content of varioustissues and lipid classes.

Monoene IsomersIn the section on absorption of fatty acid isomers, it was concluded thatabsorption of monoene isomers and un-isomerized fatty acids was similar.Many experiments have provided data that shows that after absorption, selec­tive incorporation and exclusion of isomeric fats into individual lipid classesoccur. The accepted concept is that metabolism and incorporation of eachindividual isomer depends on its double-bond configuration and position.

Of the various tissues, adipose tissue most nearly reflects diet composition(40, 171). The trans content of various tissue lipids from rats fed 10% (byweight) hydrogenated sunflower oil (32% trans) for 40 days was as follows:adipose, 9.4%; heart, 7.9%; liver, 7.3%; kidney, 3.0%; brain, 2.3% (20). Thisdata illustrates the variation in distribution of trans fatty acids in tissues.Researchers have reported a specificity for incorporation of trans fatty acidsinto lipoprotein lipid classes (45, 184, 193). For example, the data in Table 2suggest discrimination against incorporation of trans isomers into lipoproteinCE and PL fractions relative to TG. In related studies, 9t-18: 1 was preferential­ly incorporated into the phospholipids of rat liver and adrenal tissues, and theLDL and HDL lipoprotein PL fractions contained higher percentages of 9t-18: 1with no preference for VLDL compared to 9c-18: 1 (75, 186), but the lipopro­tein TG and CE fractions contained less 9t- than 9c-18: I . Thus the trans contentof the lipoprotein lipid classes depends upon which lipoprotein fraction is

Table 2 Distribution of trans fatty acids into lipoprotein lipid classes

Trans (o/c) in lipid classAnimal Lipoproteinmodel fraction TG CE PL FFA Reference

Rata VLDL 18.6 7.8 9.1 10.2 45LDL 14.0 7.6 6.0 14.9HDL 15.3 8.8 19.0 2.2

Ratb VLDL 16.9 16.4 8.5 193LDL 16.2 9.8 6.2HDL 10.1 2.0 Il.l

RabbitC VLDL 15.6 6.3 13.7 184LDL 14.1 8.4 10.9HDL 9.8 7.5 12.7

'20% by weight HSBO (48% trails) fed for 105 days.b25% by weight HSBO (44.5% trans) fed for 60 days.C20% by weight hydrogenated olive oil (37.6% trails) fed for 21 days.

ISOMERIC FATTY ACIDS 355

analyzed, but results are not consistent enough to permit general conclusions.Lipoprotein lipid fatty acids are sensitive to sampling and experimental condi­tions, and trans content might be expected to vary significantly for samplesfrom fed and fasted rats (193).

For rat brain PC, 67% of the total 9t-18: 1_ 14C incorporated was in I-acyl PCand 33% was in 2-acyl PC (104). Rat placenta did not discriminate for 9t­compared to 9c-18:1- 14C, but fetal lipid contained four times more 9t- than9c-18: 1 (145). Enrichment of9t-18: I in fetal lipid was not the result of selectiveincorporation into the PC-I position, because equal amounts of9t-18:1 wereincorporated into PC-I and PC-2. In contrast, research from the same labora­tory reported that fetal livers from progeny of rats fed hydrogenated corn oil(52% trans) contained 2.5% trans compared to 5% trans in the maternalplasma, and no trans fatty acids were detected in fetal heart or brain lipids(141). Related data for distribution of 8t- and 12t-18:1 in egg yolk lipidsshowed selective incorporation into the PC-l position (57, 120, 121).

These and other data (48, 95, 170, 227, 232) indicate that the trans fatty acidcontent of tissues depends on tissue, lipid class, total fat in diet, trans content ofdiet, and probably on duration of feeding, species, and fatty acid compositionof diet. The position and configuration of the double bond also have importanteffects on the incorporation and distribution of cis and trans positional isomerspresent in hydrogenated fats and oils. In fact, the trans fatty acid content oftissues is an underestimation of the total isomeric fatty acid content, and basedon in vitro and in vivo evidence, the cis fatty acid isomers are as important asthe trans isomers.

As an example, the relative levels of cis and trans positional 18: I isomersincorporated into heart, adrenal, adipose, and liver lipids were determined forrats fed HSBO containing 12.3% trans (171). Adrenal and adipose werenonselective for individual trans isomers, but heart and liver were selective fortrans isomers. For heart and liver, the 14t-18:1 percentage was three timeshigher, II c-18: I was two times higher, and 12t-18: I was three times higherthan in the diet. The ratio of 12c-, 13c-, and 13t-18:1 percentages in thesetissues were approximately the same as in the diet, and the ratio forthe 8t-, 9t-,and IOc-18: I percentages was lower. Only one fourth as much IOc-18: I wasfound in liver lipids, and the 10t- and I It-18: I were almost totally excluded.

In the most exhaustive rat study to date, Wood et al (227) fed partiallyhydrogenated safflower oil (51 % trans) and determined the distribution of cisand trans positional 18: I isomers in TO, PC, PE, PS, and phosphatidylinositol(PI) from kidney, muscle, heart, liver, lung, spleen, and adipose tissue. Inaddition, liver PL were analyzed for cis and trans positional 18: I isomerdistribution in the I-acyl and 2-acyl positions of PC and PE. Varying levels ofselective incorporation and exclusion of each specific isomer were reported.The more notable results were the very strong exclusion of 1Oc-18: I from liver

356 EMKEN

CE, TG, and PL and of IOt-18:1 from PL. The 12t-18:1 isomer was concen­trated in PC and PE. This study also showed selective incorporation of the 8t­and 9t-18: I into tissue TG, exclusion of JOc-18: I, and selective incorporationof 8t-, 9t-, 12t-, 13t-, 14t-, llc-, 12c-, 13c-, and 14c-18:1 into many of thevarious tissue organ PE, PC, and SM fractions. For rat liver phosphatidylcho­line, preferential acylation of the one position occurred with 11t-, 12t-, 13t-,and 14t-18: 1 and 8c-, llc-, IOc-, and 14c-18: 1 isomers. Preferential acylationof the 2-acyl PC position occurred with 9t-, JOt-, and 9c-18:1. For rat liverphosphatidylethanolamine, acylation of the one position was preferred with12t-, 11c-, 12c-, and 13c-18: 1, and acylation of the two position was preferredfor the lOt-and 9c-18: 1 isomers. Differences in PC and PE acyl specificity forthe other positional isomers were small. For the IOc-18:1 isomer, only verysmall amounts were incorporated, which indicated strong exclusion for bothacyl positions. In vivo rat studies with diets containing 6c-18: 1 and 8c-18: 1showed that isomers were incorporated primarily into the I-acyl position ofliver PC and PE at the expense of saturated fats and into adipose TG at theexpense of 9c-18:1 (94).

Analysis of positional 16: 1 isomers in rat tissue TG identified the 7t- to14t-16: 1 isomers. The percentages for the 8t-, 9t-, 10t-, and 11 t-16: 1 isomerswere approximately 45, 19, 12, and II % respectively, depending on tissuesource and indicated preferential chain shortening or partial degradation of thecorresponding t-18:1 positional isomers. In contrast, chain shortening of thec-18: 1 positional isomers appears to be of minor importance based on the lowlevels of c-16:1 positional isomers.

The incorporation of stable isotope-labeled monoenoic isomers into humanplasma lipids has been reported for the 9t-, 12t-, 12c-, 13t-, and 13c-18:1isomers relative to 9c-18:1 (52,55,56,58,59). Discrimination against ester­ification of cholesterol with the 12t- and 13t-18: 1 isomers was nearly absolute,with moderate discrimination observed for the other isomers. Selective acyla­tion of the one position of PC was observed for all the isomers. Negativeselection or discrimination against acylation of the 2-acyl position of PC wasfound for all the isomers except 12c-18: 1, which is preferentially incorporatedinto the 2-acyl PC position. Incorporation of both the 13c- and 13t-18: 1 isomersinto the various other phospholipids was negative in contrast to the positiveselective incorporation of the 9t-18: 1 isomer. These data clearly indicate thatenzyme selectivities are different for each isomer and depend on both theposition and the configuration of the double bond. Comparison of data onhumans and on rats indicates a considerable difference between species inmetabolism of these isomers.

Turnover of the fatty acid isomers in human plasma lipids was not signifi­cantly different from that of 9c-18: 1, and it is consistent with data for theisomeric fatty acid content of human tissues and with data from rats fed

ISOMERIC FATTY ACIDS 357

hydrogenated fats and then switched to diets containing nonhydrogenated oils.Similar data have been reported for disappearance of trans fatty acids fromhuman milk lipids (105). Turnover of trans fatty acids in nerve PL of rats wasslower than for heart and liver PL. Also, the rate ofdepletion of trans fatty acidsin nerve PL was 50% faster when consumption of trans fatty acid isomersoccurred after weaning rather than before (123).

Diene IsomersData on incorporation and distribution of diene isomers into tissue lipids aremainly limited to the geometrical isomers of c,c-18:2. The t,t-18:2 content intissues from rats fed diets containing 5% (by weight) of a 1: 1 mixture of c,c­and t,t-18:2 for 12 weeks was heart, 2.8%; kidney, 7.1 %; lung, 1.0%; adipose,1.6% (108,231). These levels oft,t-18:2 are comparable to those in rats fed adiet containing 5% (by weight) of a mixture of 20.8% 9t-18: 1,47.5% t,t-18:2,21.3% 16:0, and 9.5% 18:0. Even in the absence of dietary c,c-18:2, thet,t-18:2 content of liver, heart, kidney, and serum lipids ranged from only 2.1to 7.3% (204). Similarly, serum lipids from rabbits fed 6 g oft,t-18:2 per dayfor 84 days contained only 2.1-2.5% t,t-18:2 in the various PL fractions, butthe t,t-18:2 contents were 11 % for TG and 13% for CE (218). These levels oft,t-18:2 incorporation are low in view of the amounts in the diet. The datasuggest a strong exclusion for incorporation of t,t-18:2, especially into PL.

In contrast to the rat data, studies of the laying hen using t,t-18:2-3H andc,c-18:2- 14C showed a slightly lower incorporation oft,t-18:2 into egg CE andno difference between the incorporation of radioisotope-labeled t,t- and c,c­18:2 into egg neutral lipid and PL (122).

The use of 14C-Iabeled t,t-18:2 to monitor its incorporation into rat brain,liver, and fetal lipids has resulted in some interesting observations. Studies onpositional distribution of t,t-18:2 into rat brain phosphatidylcholine are contra­dictory. Using t,t-18:2- 14C, 90% of the 14C label was found in the 2-acylposition of PC (104), but analysis of brain PC and PE from rats fed unlabeledt,t-18:2 indicates a preferential distribution into the I-acyl PC position, whilec,t-18:2 was preferentially incorporated into the 2-acyl PC position (36, 164).

These latter results are consistent with liver data from EFAD rats fedmixtures of t,t- plus c,c-18:2 and t,t-18:2 plus c,c,c-18:3. In this study, thet,t-18:2 content of liver I-acyl PC was four times higher than that of2-acyl PC(188).

However, the opposite results were reported for rat fetal PC that usedt,t-18:2- 14C (146). In this study, 80% of the t,t-18:2- 14C was incorporated intothe 2-acyl position, which was similar to the distribution observed for c ,c-18:2­14c. In a related study, the levels of t,t-18:2 14C incorporated into rat fetal andplacenta lipids were, respectively, three and six times higher than the levels ofc,c-18:2 (145). The main reason for this difference probably was the higher

358 EMKEN

utilization of c,c-18:2- 14C by the maternal tissue, which lowered the amountavailable for transport across the placenta.

The observation that t-18: 1 positional isomers are desaturated to c,t- andc,c-18:2 positional isomers by rat liver microsomes provided an opportunity toobtain data on the distribution of these in situ biosynthesized diene isomers intothe various microsomal lipid classes (175). The pattern for incorporation ofpositional dienoic acid isomers was complex, and specific isomers did notfollow any particular pattern. In general, the c,c-18:2 isomers were enriched inthe PL fraction, and c,t-18:2 isomers were selectively esterified into thecholesterol ester fraction.

IN VITRO SPECIFICITIES OF ENZYME SYSTEMS FORISOMERIC FATS

Several of the enzymes involved in lipid metabolism have been investigated byin vitro techniques to determine specific enzyme activity for individual fattyacid isomers. The data reviewed in this section complement in vivo data byproviding nutrition-related metabolic and biochemical information that isotherwise difficult to obtain.

As discussed in the previous section, in vivo distribution and incorporationof dietary fat into tissue lipids depend on many factors and involve interrelatedenzyme reactions, including enzyme selectivity for specific substrates. Incontrast, the distribution of isomeric fats in vivo is determined by selectiverecognition of the unsaturated isomeric fatty acid structure by specific enzymesor enzymatic systems, but other factors also can be involved.

LipasesAs noted above, in vitro hydrolysis of triglycerides containing c-18: 1 positionalisomers by pancreatic lipase is nonspecific for isomers with the double bondbetween the 8 to 15 carbons (101). Animal and human absorption data alsosuggest that there is little if any difference in the hydrolysis of triglyceridescontaining isomeric fats in hydrogenated oils. In contrast, phospholipase isknown to hydrolyze fatty acids selectively at the 2-acyl position of phospholip­ids, but there is no evidence for selective deacylation based on geometric orpositional configuration of the double bond.

Acyl CoA Synthetase

Many lipid enzyme reactions require that long-chain fatty acids first be acti­vated by formation of the acyl CoA esters. Reaction rates for formation of theacyl CoA derivative by mitochondria and microsomal preparations have beenreported for the .:l4 through .:l17 positional isomers of c-18:1 (129), t-18:1(130), and 16:0/t,t-18:2/c,c-18:2 (128). The reaction rates for the trans fatty

9c-18:1 IOddl

.£3 p-O,idation~- Cycles

9Ce-:;C=C,C-D-SCoA

H H 0

~merase

ISOMERIC FATTY ACIDS 359

acid isomers were higher than for the cis isomers, and the rates for theeven-numbered trans positional isomers were greater than for the adjacentodd-numbered trans isomers. For both cis and trans positional 18: I isomers,rates were lowest when the double bond was near the center of the acyl chain,and the lowest rate was for the JOc-18: I isomer. Rates for acyl CoA esterformation for t,t- and c,c-18: 1 were not significantly different (120). Acyl CoAester formation rates were sensitive to temperature, fatty acid/protein ratios,detergents, and pH, which suggests that in vitro rates may be quite differentfrom in vivo rates. The kinetics for acyl CoA synthetase activation of c,c-18:2w5, w7, w8, and w9 isomers indicate the largest enzyme specificity was for the9c, 12c structure, with the ~9c bond identified as the main determinant of acylCoA synthetase specificity (202).

OxidationOxidation of fatty acyl CoA ester isomers involves severall3-oxidation cyclesuntil a double bond is encountered (see Figure 1). If the fatty acid double bondis originally in an odd-numbered position, it is then conjugated with thecarbonyI group by a ~3-~2 enoyl-CoA isomerase. For even-numbered position­al isomers the enoyl-CoA isomerase is not needed; but, for cis position-

91-18:1 IOddl

~ 3 p-O,idation~CyeleS

H\ 9

ce-c=c,C-D- SCoA

H 0

~merasa

H\9

es-C=C,D-SCoAHO

'\. Hydratase

"' ?H

~R-CH::_~~:~:~sL~COA ~imerase

Hydratesa ! \10C7-r-CHz-~-SCoA

Oehydrogenase OH 0

H "" Hydratase\ '0 " '0

C7-C=C\-~-SCOA R-C-CHz-C-SCoA C7-}=C,~-SCOA

/"".:~ ~.,! H H HO~p'O'idetionI Cycles

eye as R-C-SCoA + CHJ-C-SCoA

101-18:1 IE.Yen) H H 10c-18:1IEven)

Figure I 13-0xidation of cis and trans octadecenoic acid positional isomers containing odd· andeven-numbered double bonds.

360 EMKEN

al isomers, a 3-hydroxy acyl-CoA epimerase is required to convert the D-3­hydroxy isomer to the L-3-hydroxy isomer. The remainder of the 13-oxidationsteps are the same for all isomers.

In vitro oxidation of the ~4 to ~ 16 positional cis and trans 18: I isomers byrat heart and liver mitochondria has been followed by measurement of oxygenuptake (125). Based on oxygen uptake, even-numbered cis-18: I isomers wereoxidized more slowly than the adjacent odd-numbered cis isomers, and mosteven-numbered t-18: I isomers were oxidized more rapidly than adjacent odd­numbered trans isomers. The t-18:1 isomers also were oxidized more slowlythan their respective cis isomers. Explanations suggested for the slower oxida­tion rates include slower rates for CoA ester formation, slower transfer throughcell membranes, and differences in reaction rates for the steps involving enoylisomerase and hydroxy acyl CoA epimerase. However, various in vitro inves­tigations have not provided conclusive evidence that any of these specific stepsis the rate-controlling reaction.

Several investigators have employed" radioisotope-labeled fatty acid sub­strates to compare in vivo oxidation of various isomers to fatty acids present innonhydrogenated fats and oils. The in vitro oxidation rate (formation of CO2plus soluble products) by rat liver mitochondria for uniformly 14C-Iabeled 9c­and 9t-18: I was 20% lower for the 9t-18: I isomer (7). In contrast, it was notedthat oxidation rates based on 14C02 formation of 14C-carboxy-Iabeled 9c- and9t-18:1 were nearly identical. In other studies, rat heart perfused with fattyacids complexed with albumin removed 9c, 12c-18:2 from the perfusate morerapidly than 9t-18: I and less rapidly than 9c-18: I (223). Respiratory activity ofheart mitochondria was lower when rats were fed HSBO containing 48% t-18: Ifor six weeks (95). Oxidation rates for 14C-Iabeled t,t-; c,t-; t,c-; and c,c-18:2isomers injected into developed rat brain were similar (36). All of the in vitrodata based on 14C02 release and O2 uptake are at least qualitatively in agree­ment with in vivo rat data, which also showed slower oxidation rates for9t-18:1. For example, in vivo oxidation of9c-18:1 was 8-10% greaterthan of9t-18:1 (8). However, male rat heart homogenates recently were reported tooxidize 9c- and 9t-18: I with equal efficiency, in contrast to female rat hearthomogenates, which oxidize 9t-18:1 faster than 9c-18:1. In addition, heartoxidation rates for rats pre-fed diets containing trans isomers were substantiallyhigher for both 9c- and 9t-18:1 (141).

Recently, peroxisomal oxidation has been estimated to be responsible for10-50% of the fatty acid oxidized by the liver (157). Peroxisome oxidationrates for t-16:1 and t-18:1 were reported to be higher than for the c-16:1 andt-18: I isomers, in comparison to 13-oxidation rates by rat liver mitochondria,where the rates for the trans isomers were about one half those of the cis fattyacids.

ISOMERIC FAITY ACIDS 36I

Other studies have reported increased peroxisomal oxidation levels for ratsfed HSBO, trans fatty acids, hydrogenated marine oils, and high-fat diets (33,97, 149, 206). In contrast, a 26-week in vivo rat study using hydrogenatedmarine oil, rapeseed oil, and peanut oil diets showed no increase in peroxiso­mal oxidation rates (91).

Retroconversion

In vitro data have provided evidence that chain shortening or retroconversionoccurs for a number of isomeric fatty acids and to a greater extent than for9c-18: 1 or 9c, l2c-18:2. Examples of retroconversion reactions observed in ratheart perfused with various positional 18: 1 isomers are: 8t- and 8c-18: 1-14: 1;9t-18: 1-5t-14:1; lOc-18: 1-12:1; 1It-18: l-5t-12: 1. Conversion of 11c-18: 1and lOt-18: 1 to shorter-chain-length fatty acid products was not detected (223).Utilization of 9t-18:1-10 14C by mouse sciatic nerve cells grown in tissueculture also resulted in five times more radioactivity in the 16: 1 fraction thanwith 9c-18:1-10 14C (21). More 14:1 was also produced from 9t-18:1-IQ14C.

These in vitro data are consistent with in vivo rat fetal phospholipid data thatreported 30% more 14C in 16:1 from 9t-18:l when dams were fed labeled9t-18:1 and 9c-18:1 (145). Wood (225) identified positional 16:1 isomers intissues from rats fed hydrogenated safflower oil acids. In addition, increasedlevels of 16: 1 in many tissue lipids have been observed in in vivo studies where18: 1 isomers or HSBO were fed, and this suggests that chain shortening of 18: 1isomers to 16:1 isomers is of possible nutritional importance. The retroconver­sion data for isomeric fats indicate that the f3-oxidation cycle is interrupted orslowed after 1- to 3-acyl CoA residues are removed and that the chain­shortened fatty acid is ejected from the cycle.

Phospholipid Acyl transferase

The preferential incorporation of trans fatty acid isomers into the I-acylposition of phospholipids has been observed in many in vivo studies and isgenerally considered evidence of phospholipid acyl transferase specificity. Arecent example is taken from a study of rats fed a diet containing 18.5% (byweight) HSBO composed of 12.3% t-18:1 and 25% 18:2. The percentage oft-18: 1 isomers incorporated was 8-20 times higher in I-acyl PC than in 2-acy1PC, depending on the tissue type analyzed (170). Data from rats fed HSFOcontaining known amounts of individual positional cis and trans 18: 1 isomersshow a general preferential incorporation of these isomers into the I-acylposition of phospholipid classes from a number of organs (140).

The in vivo data are in general agreement with in vitro acyl CoA:phospholi­pid acyl transferase rates reported for a series of.!l2 to .!l17 cis and trans 18:1

362 EMKEN

isomers (155, 173). For the series of t-18:1 positional isomers, the acyltransferase rates for esterification of the I-acyl PC position were much moresensitive to double-bond position than the 2-acyl PC rates, and I-acyl PC rateswere significantly higher. Acyl transferase rates for the lOt- and 14t-18:1isomers were very low compared to their adjacent odd-numbered positionalisomers. For I-acyl PC, the highest rates were obtained for the 8-, 9-, 11-, and13t-18:1 isomers. Within the series of cis positional 18:1 isomers, the acyltransferase rates were higher for the 5-, 8-, 10-, 12-, and 15c-18: I isomers thanfor isomers with double bonds adjacent to these positions. The highest reactionrate was found for 12c-18: I, which was 20% higher than for 8c-18: 1, thesecond highest rate. Surprisingly, the rate for 12c-18: I was about nine timesthat of the rate for 9c-18: 1. With purified rat liver microsomes, the rate foracylation of the 2-position of PC with 9c-18: I was 2.4 times higher than thesecond highest rate, which was for 12c-18: 1. In comparison, rates with crudemicrosome preparations for 12c-18: I were 3.3 times higher than for 9c-18: 1,the second highest rate. These data suggest that the 12c double bond has specialbiological significance and is one factor responsible for the observed selectiveincorporation of 9c, 12c-18:2 into the 2-acyl position of phosphatidylcholine.

Others also have reported selective acyl transferase activity for specificpositional isomers. Rat brain acyl transferase selectively esterified the II c-18: Iisomer at the I-position of PC compared to 9c-18: I, which was selectivelyincorporated into the 2-acyl PC position (147). Similarly, perfused chickenliver selectively incorporated 18:0, 9t-, and I It-18: I into the I-acyl position ofPE and PC relative to 9c and Ilc-18:1, which were selectively placed in the2-acyl positions (18).

The relative activities of rat liver microsome acyl transferase for cis and transisomers of various chain lengths were 100 for 9c-18: I; 76 for 9c-16: I; 45 for9t- i6: I; 32 for 9t-18: I; 12.5 for Ilc-20: I; 5.3 for Ilt-20: I; and 0 for 13c- and13t-22:1 with 1-palmitylglycerol 3-phosphate as a cosubstrate (136). Thus,both chain length and double-bond configuration influence selectivity.

The relative incorporation of tritium-labeled 115 to illS cis 18: 1 isomers intorat liver mitochondria lipids has been compared to that of 9c_18:1_ 14C (194).All the tritium-labeled isomers were selectively incorporated into total phos­pholipids compared to 9c-18:1- 14C. In general, the odd-numbered isomerswere incorporated at lower levels than the adjacent even-numbered isomers,and amounts incorporated increased when the location of the double bond wascloser to the carboxyl and methyl ends of the fatty acid chain. No selectiveincorporation into the 2-acyl position of PC was found for the isomers, andstrong discrimination was found for 14c- and 15c-18: I (194). Many of these invitro and in vivo results are not consistent, and there is much to be learned aboutthe factors that determine the activity and selectivity of acyl transferase formonounsaturated isomers.

ISOMERIC FATTY ACIDS 363

Compared to rates for the 18:1 isomers, the acyl transferase rates forincorporation of the diunsaturated trans isomers into rat, cow, and human redblood cell phosphatidylcholine were quite different (215). Acylation of theI-acyl position of PC by human red blood cell stromata was barely detectable.For the 2-acyl PC position, the acyl transfer rates (mol/min/mg protein)catalyzed by human stromata were 0.7 for 18:0; 5.3 for 9c-18: I; 3.7 for 9t-18: I ;2.4 fort,t-18:2; 3.9 for c,t-18:2; 11.4 for t,c-18:2; 14.0 forc,c-18:2; and 8.1 for20:4. The difference in rates between the 9c,12t- and 9t-,12c-18:2 isomersreflects the importance of the 12c double bond in acyl transferase reactions.Rates for rat stromata were similar, except that values for the 9t- and 9c-18: Iwere reversed in comparison to the human data.

The acyl transferase rates for esterification of 1- and 2-acyl PE and PC alsohave been reported for the entire series (2,5- to 14,17-) of methylene­interrupted c,c-18:2 positional isomers (174). All of the acyl transfer reactionrates for the 2-acyl PC position were greater than or equal to the rates for theI-acyl PC position, which is consistent with the preference for placement ofpolyunsaturated fats at the 2-acyl PC position. Highest activities were obtainedfor the 5,8-; 11 ,4-; 9,12-; and 13,16-18:2 isomers relative to the isomers withadjacent double bond systems. With 2-acyl PC as the substrate, highest activi­ties werefor the 8,11-; 12,15-; 13,16-; and 14,17-18:2 isomers. Reaction rateswere low for all isomers when I-acyl phosphatidylethanolamine was thesubstrate; with 2-acyl phosphatidylethanolamine as the substrate, high activi­ties was obtained forthe 8,11-; 10,13-; II, 14-; 12,15-; 13,16-, and 14,17-18:2isomers. In vivo positional specificity for incorporation of 9c.15c-, and12c,15c-18:2 isomers into rat liver PC has also been reported (198). The9c, 15c-18:2 isomers were almost exclusively esterified at the 2-acyl position ofPC, while 12c,15c-18:2 was equally distributed between the 1- and 2-acylpositions; this agrees very closely with in vitro data. Both reaction rates and invivo data indicate that both double-bond positions in 18:2 are involved incontrolling the reaction rates, and that the entire methylene-interrupted dienesystem is an important structural feature. The fact that the highest rates were notobtained for 9,12-18:2 is unexpected, given the recognized biological import­ance of linoleic acid.

Cholesterol Esterase and HydrolaseBiosynthesis of cholesteryl ester is catalyzed by acyl CoA:cholesterol acyltransferase (ACAT) and lecithin:cholesterol acyl transferase (LCAT). Ole­oyl CoA is the best substrate for ACAT, and LCAT favors the formationof polyunsaturated cholesteryl esters (67, 68, 199). In rats, the predomi­nant pathway for cholesteryl esters synthesis involves ACAT; in humans,LCAT is the primary enzyme responsible for plasma cholesterol ester forma­tion.

364 EMKEN

Relative reaction rates have been reported for a pancreatic cholesterolesterase (source unknown) using a number of fatty acid substrates (112). Bothfatty acid chain length and double-bond configuration are reported to havesignificant effects on the reaction rates. Relative to 9c-18: 1, the reaction ratesfor 9t-18: 1, lIt-18: 1, and llc-18: 1 were 0.5, and for 9t, 12-18:2 it was 0.7. Forcomparison, the rate for· stearic acid was 0.2 and for lauric acid, 0.8.

With ACAT from rat liver microsomes , the esterification rates indicatedstrong preference for acyl CoA esters of 9c-18: 1 (192). The rates for 1It-18: 1,9t-18:1, 9c,12c-, and 9t,12t-18:2 were approximately 33% of the 9c-18:1 rateand 80% for 11c-18:!. These data plus in vivo data for ACAT and in vivohuman CE data for LCAT suggest that both enzymes are very sensitive todouble-bond position and configuration.

In contrast, rat liver cholesterol hydrolase activity for cholesterol esterscontaining the cis-18: 1 positional (Li2 to Li 17) isomers was relatively nonspeci­fic (69). Maximum activity was reported for the 9c double bond, and activitygradually decreased as the double-bond position was moved towards either endof the fatty acid acyl chain. Hydrolysis rates for both 9t-18: 1 and 9t,12t-18:2were intermediate between those of 9c-18:l or 9c,12c-18:2 and 18:0 or 16:0(191).

Desaturases and ElongasesDietary fatty acids, including isomeric fatty acids, are interconverted bydesaturation and elongation to yield a number of fatty acid products. Theenzymes and reaction pathways for interconversions of many of the noni­someric fatty acids have been reviewed (25, 196, 197). Generally, mammaliansystems cannot introduce a new double bond between a double bond in the 9position and the methyl end of the fatty acid chain, although a few species havethis capability to a limited extent (76). The lack of significant Li12 desaturaseactivity accounts for the requirement that diets contain 9c, 12c-18:2 and for itsdesignation as an essential fatty acid.

In addition to undergoing desaturation and elongation, isomeric fatty acidsalso function as competitive inhibitors for desaturase and elongase activity. Invivo evidence for inhibition of desaturation and elongation of 18:2 by t,t-18:2has been reported that shows that t,t-18:2 suppresses 20:4 levels in rat tissues(9,36, 37, 108, 165). Depression of Li6 and Li9 desaturase activity by t,t-18:2and HSBO has also been reported for rat brain (37, 85), and desaturase­elongase rates for t,t-18:2 and c,t(t,c)-18:2 were about six times slower thanfor c,c-18:2 (36). Conversion of c,t-18:2 to 20:4 occurs readily, but 9t,12c­18:2 conversion to 20:4 is very slow (9, 19,81, 166). The t,t-18:2 isomeralso inhibits desaturation-elongation of 18:1 and 16:1 to 20:3w9 and 20:4w7(166).

ISOMERIC FATTY ACIDS 365

In vivo results suggested that HSBO diets depressed 66 and 69 desaturateactivity in rat liver, but not 65 desaturase activity (85). Rat liver microsomedata indicated the degree of inhibition for each of the 63 to 616t-18: 1 isomerson desaturation of 16:0, 18:2, and 20:3 depended on double-bond position(134). Competitive inhibitors for the 65, 66, and 69 desaturases werestrongest for the following isomers: 3-, 9-, 13-, and 15t-18: I (65 desaturase);5-,4-,7- and 15t-18:1 (66 desaturase); and 3-, 5-, 7-, lO-, 12-, and 13t-18:1(69 desaturase). Desaturation of 18:0 by rat liver microsomes was also inhib­ited by the 62-to 617c-18: 1 isomers, the 62- to 612-18: 1 acetylenic acids, andthe 6t-; 9t-; and lIt-18:1 isomers (32). However, Downing et al reported that18:1 acetylenic acids did not inhibit desaturase activity (43).

The desaturation products of various positional cis and trans 18: 1 and 18:2isomers summarized in Tables 3 and 4 are largely from rat liver microsomestudies (133, 135, 163). In comparison to c-18:1 positional isomers, t-18:1positional isomers were desaturated to both c,t- and c,c-18:2 isomers by ratliver microsome preparations. Only 69 desaturase activity for t-18: 1 positionalisomers was observed by Mahfouz et al (135), but Pollard et al (163) observeddesaturation of 8t-18: 1 at the 65-position. In addition, the octadecadienoic acidisomers formed from 611-,613-,614-, and 615t-18:1 were subsequentlydesaturated at the 66-position. Desaturation studies of the 64- to 611c-18: 1isomers by rat liver microsomes reported that the 8c and lIe were desaturatedby 65 desaturase, 9c-18:1 was desaturated by 66 desaturase, and lOc and lIewere very slowly desaturated (133, 163). The 64- to 67c-18:1 isomers werenot desaturated. In addition, a few cis and trans 16:1 and 17:1 isomers werereported to be desaturated to 16:2 and 17:2 isomers (163). Recently, conversionof the t-18:1 isomers to t,c- and c,c-18:2 isomers was confirmed by in vivostudies with rats (124). The conversion of trans monounsaturated isomers tocis, cis polyunsaturated isomers by rat liver apparently involves an isomeraseas well as a desaturase. The observation that cis and trans 18:1 positionalisomers are converted to octadecadienoic acid isomers is a factor that has notbeen considered in the interpretation of most physiological and metabolicstudies with isomeric fatty acids.

Elongation of the 64- to 615t-18:1 isomers by rat liver reported the 64-,65-,66-,613-,614-, and 615t-18: 1 isomers were not elongated and that verylow rates for elongation of 68-, 6lO-, 611-, and 612t-18:1 occurred. The 67t­and 69t-18:1 were elongated to 20:1 (103).

Previous data indicate that rats have enzyme systems capable of thebiosynthesis of a large variety of fatty acids. Nineteen 18:1 isomers, fifteen18:2 isomers, and nine 18:3 isomers identified in tissue lipids from rats on afat-free diet have been reported (182), and other similar data have beenpublished (225,226). Presumably these isomers are formed by desaturation ofendogenously synthesized 18:0. This ability to synthesize a wide range of

366 EMKEN

Table 3 Desaturation of octadecenoic acid isomers

Substrate 18:2 Product(s)(18:1 isomer) structure Reference

4t- 4t.9c- 1354c.9c- 135

5t- 5t.9c- 1635c.9c- 135

6t- 6t,9c- 1356c.9c- 135

7t- 7t.9c- 135. 163

8t- 6c.8t- 163

9t- 5c.9t- 74. 163

IOt- 5c.IOt- 163

I It- 6c.llt- 1639c.llt- 135. 1639c,llc- 135

12t- 9c.12t- 1359c.12- 135

13t- 9c,13t- 135. 1639c,13c- 1356c,l3t- 163

14t- 9c.14t- 135, 1635c.14t- 163

15t- 9c.15t- 1635c.15t- 163

4c to 7c- none 133, 163

8c- 5c,8c- 133. 163

9c- 6c.9c- 133. 163

IOc- 5c.IOc- 1337c,IOc- 133

IIc- 6c,llc- 1635c,llc- 133, 163

12c- 9c.12c- 76

13c- 6c.13c- 1639c.l3c- 163

14c- 9c,14c- 1636c,14c- 163

15c- 9c,15c- 1636c.15c- 163

ISOMERIC FATTY ACIDS 367

Table 4 Desaturation of octadecadienoic acid isomers

Substrate Structure of(18:2 isomer) desaturation product Reference

9c,llt- 6c,9c,llt-18:2 163

9c,12t- 6c,9c,12t-18:3 1635c,8c,llc,14t-20:4 19, 166

9t,12c- none 9

9t,12t- 6c,9t,12t-18:3 163

9t,12t- none 122, 166, 146

9c,13t- 6c,9c,13t-18:3 163

9c,14t- 6c,9c,14t-18:3 163

9c,15t- 6c,9c,15t-18:3 163

9c,15c- none 198

12c,15c none 198

isomers has not been demonstrated in humans, but it suggests that rats might beexpected to have a greater capability to metabolize isomeric fats from dietarysources than do humans. If this is the case, then rat data are not an accuratereflection of isomeric fatty acid metabolism in humans.

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